Composition of 5-nitrobenzoate derivatives as anti-metastatic agent that inhibits tumor cell-induced platelet aggregation

ABSTRACT

Disclosed are 5-nitrobenzoate derivatives of Formula I, 
     
       
         
         
             
             
         
       
     
     and the preparation method therefor, wherein R is referred to hydrogen (H), unsubstituted, mono-substituted, di-substituted or tri-substituted benzoyl moiety. 5-Nitrobenzoate derivatives of Formula I do not affect the platelet aggregation, possesses the inhibitory activity related to the tumor cell-induced platelet aggregation (TCIPA), and further specifically inhibits podoplanin-induced platelet aggregation. Therefore, 5-nitrobenzoates of the invention are applicable in its therapeutic use as the novel therapeutic agent in preventing tumor metastasis.

CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The application claims the benefit of Taiwan Patent Application No.101124690, filed on Jul. 9, 2012, in the Taiwan Intellectual PropertyOffice, the disclosures of which are incorporated herein in theirentirety by reference.

FIELD OF THE INVENTION

The present invention relates to a 5-nitrobenzoate derivative which isused in the therapy for cancer metastasis via the inhibition of tumorcell-induced platelet aggregation (TCIPA).

BACKGROUND OF THE INVENTION

Tumor cells can stimulate platelet activation and form the aggregationcomplex with platelets in the vascular circulation system. Thisinteraction is termed as tumor cell-induced platelet aggregation(TCIPA). The ability of tumor cells to induce platelet aggregation hasbeen proven to highly correlate with the metastatic capability ofmalignant tumor. At present, it is known that there are many factors andmechanisms involved in TCIPA. For instance, tumor cells activatecoagulation cascade via thrombin generation and induce plateletaggregation. Besides, adenosine diphosphate (ADP) release is involved inMCF-7 tumor cells-induced platelet aggregation that is relevant to theexpression of platelet surface P2Y12 receptor (Alonso-Escolano et al.,Br. J. Pharmacol. 141: 241-252, 2004). Other factors including (1)proteinases: cathepsin B and matrix metalloprotease (MMPs), (2)thromboxane A2 and prostacyclin, (3) nitric oxide (NO), (4) plateletsurface proteins (e.g. GPIb-IX-V, GPIIb/IIIa and P-selectin, etc.) andso on are involved in TCIPA (Jurasz et al., Br. J. Pharmacol. 143:819-826, 2004). Based on these results, the detail mechanism of TCIPAseems to very complicated and still be obscured. Nevertheless, theseresults highlight TCIPA as a target for development of cancertherapeutic strategies in translational medicine.

As so far, various approaches and anti-platelet agents have beenreported to inhibit TCIPA that potentially can be used as the strategiesfor treatment of tumor metastasis. For example, theanti-platelet/coagulation molecule heparin can reduce thecancer-associated thromboembolism risk. In clinic, low molecular weightheparin (LMWH) is administrated to cancer patients to inhibit factor Xaand thrombin to block platelet aggregation, despite that LMWH is nottumor-specific and LMWH overdose usually increases the bleeding risk ofcancer patients (Borsig, Progress in Molecular Biology and TranslationalScience. 93: 335-349, 2010; Lee, et al., N. Engl. J. Med. 349: 146-153,2003). A common salicylate acid drug, aspirin, inhibits cyclooxygenase(COX) and subsequently blocks thromboxane A2 (TXA2) generation andplatelet aggregation. Nevertheless, high dose aspirin does not showspecificity and does not have effects on cancer metastasis and patientprotection in clinics (Jurasz et al., Br. J. Pharmacol. 143: 819-826,2004). Recently, utilizing antibody to obstruct the interaction betweenplatelets and tumor cells serves as a promising approach to blockmetastasis. For example, anti-aggrus/podoplanin antibody has been usedto inhibit the interaction of TCIPA between the transmembrane protein(“podoplanin”) of cancer cell and C-type lectin-like receptor 2 (CLEC-2)of platelet. Antibody therapy is usually expensive and patients mightsuffer from the risk of autoantibody generation (Nakazawa et al., CancerSci. 102: 2051-2057, 2011). On the whole, the problems in the prior artlie in that the functions of platelet aggregation are still influencedsuch that tumor cells cannot be specifically inhibited and TCIPA is notefficiently inhibited.

It is therefore attempted by the applicant to deal with the abovesituation encountered in the prior art.

SUMMARY OF THE INVENTION

For overcoming the drawbacks in the prior art, a series of5-nitrobenzoate derivatives are obtained via the synthesis in theinvention, do not affect the platelet aggregation, and can effectivelyinhibit TCIPA and specifically inhibit the platelet aggregation inducedby surface protein, podoplanin, of tumor cells. Therefore,5-nitrobenzoate derivatives of the invention can be applicable as thetargeted therapy drug for inhibiting tumor cells.

The present invention provides a 5-nitrobenzoate derivative representedby formula I,

wherein R may be hydrogen (H), unsubstituted benzoyl moiety

mono-substituted benzoyl moiety

di-substituted benzoyl moiety

or tri-substituted benzoyl moiety

each of R₁, R₂, R₃, R₄, R₅ and R₆ may be but not limited to fluoride(F), chloride (Cl), bromide (Br), iodide (I) or methyl (—CH₃) group. R₁,R₂, R₃, R₄, R₅ and R₆ respectively are referred to bind to the para-,meta- or ortho-position of the benzene structure.

The 5-nitrobenzoate derivative can be prepared as a pharmaceuticalcomposition or a pharmacologically acceptable salt.

The present invention further provides a method for preparingN-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide, including steps of: (a)reacting 1-chloro-4-nitro-2-(trifluoromethyl)benzene dissolved indimethyl sulfoxide (DMSO) with sodium hydroxide (NaOH) to obtain4-nitro-2-(trifluoromethyl)phenol (compound 1); (b) reacting compound 2with tert-butyl 2-aminoethylcarbamate in a solution containing NaOH and1,4-dioxane to obtain tert-butyl2-(2-hydroxy-5-nitrobenzamido)ethylcarbamate (compound 2); (c) reactingcompound 3 with a dichloromethane (CH₂Cl₂) solution containingtrifluoric acid (TFA) to obtainN-(2-aminoethyl)-2-hydroxy-5-nitrobenzamide (compound 3); and (d)reacting compound 3 with benzoyl chloride to obtainN-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide (compound 4).

Preferably, compound 1 is obtained by an extraction with CH₂Cl₂, andcompound 2 is obtained by further an extraction with CH₂Cl₂.

The present invention further provides a method for preparing a5-nitrobenzoate derivative of formula I. The method includes: reactingcompound 3 with benzoyl chloride

mono-substituted benzoyl chloride

di-substituted benzoyl chloride

or tri-substituted benzoyl chloride

when R is correspondingly the unsubstituted benzoyl moiety,mono-substituted benzoyl moiety, di-substituted benzoyl moiety ortri-substituted benzoyl moiety, as mentioned above. Each of R₁, R₂, R₃,R₄, R₅ and R₆ may be fluoride (F), chloride (Cl), bromide (Br), iodide(I) or methyl (—CH₃) group.

The present invention further provide a method for preparing a5-nitrobenzoate derivative of formula I by using1-chloro-4-nitro-2-(trifluoromethyl)benzene as a starting material or anintermediate, wherein the substituted groups R, R₁, R₂, R₃, R₄, R₅ andR₆ of formula I are described above.

The present invention further provide a method for preparing a5-nitrobenzoate derivative of formula I by using compound 3 as astarting material or an intermediate

The present invention further provides a method of inhibiting tumorgrowth and/or platelet aggregation by administrating to a subject inneed thereof an effective amount of a 5-nitrobenzoate derivative offormula I. The subject may include but not limited to a human and amammal excluding the human.

The present invention further provides a method for blocking aninteraction between C-type lectin-like receptor 2 (CLEC-2) andpodoplanin or blocking an effect caused by the interaction therebetweenby using a 5-nitrobenzoate derivative of formula I.

The present invention further provides a method for blocking a pathwayof tumor cell-induced platelet aggregation (TCIPA) by using a5-nitrobenzoate derivative of formula I.

The term “derivative” herein is referred to that one hydrogen atom orsubstituted group of a molecule is replaced to form another molecule.The term “compound” herein is referred to that two or more than twomolecules are chemically bound to form another molecule at a certainmolar ratio (or weight ratio) under an adequate reaction condition.

The term “compound” herein can be made by the preparation methoddisclosed in the embodiments. The substituted group of a specificcompound can be replaced by other substituted groups, and thus otherderivatives can be prepared under this spirit. Therefore, the terms“5-nitrobenzoate derivative”, “compound” and “derivative” herein can bealternately used.

The term “platelet activation stimulator” herein is widely referred to areagent capable of activating platelets, including but not limited toadenosine diphosphate (ADP), collegen, thromboxane A2 analogs(9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619), Sigma,U.S.) and thrombin for promoting platelet aggregation, or A23187(calcium ion carrier, also named as calcimycin or calcium ionophore, AGScientific, U.S.) for increasing cationic ions (e.g. Ca²⁺) concentrationin cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objectives and advantages of the present invention will becomemore readily apparent to those ordinarily skilled in the art afterreviewing the following detailed descriptions and accompanying drawings.

FIGS. 1( a), 1(b), 1(c), 1(d) and 1(e) respectively depict the effect ofcompound 4 or the control compound 5 on (a) ADP-, (b) collagen-, (c)U46619-, (d) thrombin- and (e) A23187-induced wash platelet aggregationtests.

FIG. 2( a) depicts the effect of compound 4 and control compounds 5, 6and 7 on ADP-induced platelet-rich plasma aggregation test, whereingroup 1 is ADP (10 μM, control), group 2 is compound 4 (20 μM)pre-treatment+ADP (10 μM), group 3 is compound 5 (20 μM)pre-treatment+ADP (10 μM), group 4 is compound 6 (20 μM)pre-treatment+ADP (10 μM), and group 5 is compound 7 (20 μM)pre-treatment+ADP (10 μM).

FIG. 2( b) depicts the effect of compound 4 and control compounds 5, 6and 7 on collagen-induced platelet-rich plasma aggregation test, whereingroup 1 is collagen (4 μg/ml, control), group 2 is compound 4 (20 μM)pre-treatment+collagen (4 μg/ml), group 3 is compound 5 (20 μM)pre-treatment+collagen (4 μg/ml), group 4 is compound 6 (20 μM)pre-treatment+collagen (4 μg/ml), and group 5 is compound 7 (20 μM)pre-treatment+collagen (4 μg/ml).

FIG. 3( a) depicts the immunoblotting pattern showing the level ofpodoplanin of C6 tumor cell lines (including C6-LG, C6-Blood andC6-Lung).

FIG. 3( b) depicts the effect of compound 4 (20 μM) on C6-Lung tumorcell-induced platelet aggregation.

FIG. 4 depicts the activation analysis of compound 4 (20 μM) onrecombinant fusion protein (i.e. recombinant podoplanin/Fc)-inducedplatelet aggregation test.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically withreference to the following Embodiments. It is to be noted that thefollowing descriptions of preferred Embodiments of this invention arepresented herein for purpose of illustration and description only; it isnot intended to be exhaustive or to be limited to the precise formdisclosed.

Embodiment 1: Preparation ofN-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide

The preparation procedure ofN-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide is represented by thefollowing formula II.

1-Chloro-4-nitro-2-(trifluoromethyl)benzene (2.0 mL, 13.3 mmole) wasdissolved in dimethyl sulfoxide (DMSO, 12 mL), NaOH (1.6 g) wasbatchwise added at a temperature lower than 25° C., and the reactionsolution was reacted at room temperature (RT) for 8 hours. After thereaction was terminated, the pH of the reaction solution was adjusted to1.0 using concentrated HCl, and then the reaction solution was pouredinto the separatory funnel and extracted with CH₂Cl₂ for five times(each for 20 ml). The obtained CH₂Cl₂ solution was hydrated over MgSO₄and concentrated under vacuum. The obtained concentrate was subjected tothe purification of silica gel column (50 g) and eluted with the systemof CHCl₃/n-hexane (2:1) to afford compound 1 (1.85 g), yield of about67%.

Compound 1: ¹H NMR (400 MHz, CDCl₃): δ 8.48 (1H, d, J=2.4 Hz), 8.32 (1H,dd, J=8.8, 2.4 Hz), 7.14 (1H, d, J=8.8 Hz).

Compound 1 was nominated as 4-nitro-2-(trifluoromethyl)phenol.

Next, compound 1 (500 mg, 2.4 mmole) and tert-butyl2-aminoethylcarbamate (769 mg, 4.8 mmole) were transferred in a reactionbottle, 1 M aqueous NaOH solution (7.2 mmole) and dioxane (10 mL) wereadded, and then the mixture solution were heated to 100° C. and reactedfor 24 hours. After the reaction was terminated, the pH of the reactionsolution was adjusted to 1.0 using 1 N HCl solution, and then thereaction solution was poured into the separatory funnel and extractedwith CH₂Cl₂ for five times (each for 20 ml). The obtained CH₂Cl₂solution was hydrated over MgSO₄ and concentrated under vacuum. Theobtained concentrate was subjected to the purification of silica gelcolumn (50 g) and eluted with the system of CHCl₃/n-hexane (19:1) toafford compound 2 (600.0 mg), yield of about 77%.

Compound 2: ¹H NMR (400 MHz, Acetone-d6): δ 8.96 (1H, s), 8.74 (1H, d,J=2.4 Hz), 8.30 (1H, dd, J=8.8, 2.4 Hz), 7.10 (1H, d, J=8.8, 2.4 Hz),6.32 (1H, s), 3.58 (2H, m), 3.39 (2H, m).

Compound 2 was nominated as tert-butyl2-(2-hydroxy-5-nitrobenzamido)ethylcarbamate.

Next, compound 2 (600 mg) was installed in the reaction bottle, theCH₂Cl₂ solution containing 20% TFA was added, and the reaction solutionwas reacted at RT for 2 hours to form the reaction mixture. After thereaction was terminated, the reaction mixture was concentrated undervacuum. The obtained concentrate was subjected to the purification onsilica gel column (45 g) and eluted using the system of CHCl₃/n-hexane(4:1) to afford compound 3 (390 mg), yield of about 95%.

Compound 3: ¹H NMR (400 MHz, CD₃OD): δ 8.79 (1H, d, J=1.6 Hz), 8.21 (1H,dd, J=9.2, 1.6 Hz), 7.01 (1H, d, J=9.2 Hz), 3.72 (2H, m), 3.21 (2H, m).

Compound 3 was nominated as N-(2-aminoethyl)-2-hydroxy-5-nitrobenzamide.

Subsequently, compound 3 (390 mg) was dissolved in 2 N NaOH solution (10ml) and reacted with benzoyl chloride at RT for 16 hours, and then themixture concentrated under vacuum after the reaction was terminated. Theresidue was subjected to the purification on silica gel column (50 g)and eluted using the system of CHCl₃/n-hexane (30:1) to afford compound4 (325 mg), yield of about 57%.

Compound 4: ¹H NMR (400 MHz, C₅D₅N): δ 12.65 (1H, s), 10.14 (1H, s),9.48 (1H, s), 9.09 (1H, s), 8.11 (3H, m), 7.40 (3H, m), 7.05 (1H, J=8.8Hz), 3.95 (4H, m). ESI-MS m/z 330 (100) [M+H]⁺, 352 (32) [M+Na]⁺.HRESI-MS m/z 352.0911 (calc.: 352.0909; C₁₆H₁₅N₃O₅Na).

Compound 4 was nominated asN-(2-benzamidoethyl)-2-hydroxy-5-nitrobenzamide.

Embodiment 2: Preparation of Other 5-nitrobenzoate Derivatives

For affording other 5-nitrobenzoate derivatives, compound 3 may bereacted with benzoyl chlorides bound with a various of substitutedgroups, such as mono-substituted benzoyl chloride, di-substitutedbenzoyl chloride or tri-substituted benzoyl chloride

respectively), and each of R₁, R₂, R₃, R₄, R₅ and R₆ may be fluoride,chloride, bromide, iodide or methyl group, and R₁ to R₆ may be bound tothe para-, meta- or ortho-position of the benzoyl moiety. That is, thebenzoyl moiety of the prepared compound 4 may be substituted asmono-substituted benzoyl moiety, di-substituted benzoyl moiety ortri-substituted benzoyl moeity

respectively).

Embodiment 3: Preparation of 4-O-benzoyl-3-methoxy-β-nitrostyrene(compound 5)

trans-4-Hydroxyl 3-methoxyl-β-nitrostyrene and benzoyl chloride, weredissolved in a mixture solution of pyridine (1 ml) and CH₂Cl₂ (10 ml),and reacted at RT for 24 hours. After the removal of solvent, theobtained concentrate was subjected to the purification of silica gelcolumn (90 g) and eluted with the system of n-hexane/acetone (3:1) toafford 4-O-benzoyl-3-methoxy-β-nitrostyrene (compound 5; as representedby formula III).

Compound 5: ¹H NMR (400 MHz, CDCl₃): δ 8.25 (1H, s), 8.24 (1H, s), 8.02(1H, d, J=13.6 Hz), 7.68(1H, d, J=7.6 Hz), 7.61(1H, d, J=13.6 Hz),7.56(1H, d, J=7.6 Hz), 7.55(1H, d, J=7.6 Hz), 7.28(1H, d, J=8.2 Hz),7.24(1H, d, J=8.2 Hz), 7.17 (1H, d , J=1.2 Hz), 3.88 (3H, s). ESI-MS m/z322 (100) [M+Na]⁺.

Compound 5 was nominated as 4-O-benzoyl-3-methoxy-β-nitrostyrene).

Embodiment 4: Preparation of 4-O-nicotinoyl-3-methoxy-β-nitrostyrene(compound 6)

trans-4-Hydroxyl 3-methoxyl-β-nitrostyrene and nicotinoyl chloridehydrochloride, were dissolved in a mixture solution of pyridine (1 ml)and CH₂Cl₂ (10 ml), and reacted at RT for 16 hours. After the removal ofsolvent, the residue was subjected to the purification of silica gelcolumn (60 g) and eluted with the system of n-hexane/CHCl₃ (1:3) toafford 4-O-nicotinoyl-3-methoxy-β-nitrostyrene (compound 6; asrepresented by formula IV).

Compound 6: ¹H NMR (400 MHz, CDCl₃): δ 9.40 (1H, br.s), 8.87 (1H, d,J=4.8 Hz), 8.45 (1H, d, J=8.4 Hz), 8.00 (1H, d, J=13.6 Hz), 7.59 (1H, d,J=8.0, 2.0 Hz), 7.49 (1H, dd, J=4.8, 8.0 Hz), 7.24 (2H, m), 7.15(1H, s),3.88 (3H, s).

Compound 6 was nominated as 4-O-nicotinoyl-3-methoxy-β-nitrostyrene.

Embodiment 5: Preparation of4-O-(2,4-dichlorobenzoyl)-3-methoxy-β-nitrostyrene (compound 7)

trans-4-Hydroxyl 3-methoxyl-β-nitrostyrene and 2,4-dichlorobenzoylchloride, were dissolved in a mixture solution of pyridine (1 ml) andCH₂Cl₂ (10 ml), and reacted at RT for 24 hours. After the removal ofsolvent, the residue was subjected to the purification of (a) silica gelcolumn (100 g) and eluted with the system of n-hexane/CHCl₃ (1:2) and(b) silica gel column (60 g) and eluted with the system ofn-hexane/acetone (4:1) twice to afford4-O-(2,4-dichlorobenzoyl)-3-methoxy-β-nitrostyrene (compound 7; asrepresented by formula V).

Compound 7: ¹H NMR (400 MHz, CDCl₃): δ 8.07 (1H, d, J=8.0 Hz), 7.98 (1H,d, J=13.6 Hz), 7.57 (1H, d, J=13.6 Hz), 7.55 (1H, d, J=2.0 Hz), 7.39(1H, dd, J=8.0, 2.0 Hz), 7.25 (1H, d, J=8.0 Hz), 7.20 (1H, dd, J=8.0,1.2 Hz), 7.14 (1H, d, J=1.2 Hz), 3.88 (3H, s).

Compound 7 was nominated as4-O-(2,4-dichlorobenzoyl)-3-methoxy-β-nitrostyrene.

Experiment 1: Preparation of Human Platelets

The venous blood was collected from 18 to 35 year-old healthy volunteerdonors (who didn't take any anti-platelet medicine or otheranti-inflammation medicine within two weeks before blood draw),sufficiently mixed with anticoagulant (venous blood:anticoagulant=9:1),and then centrifuged at 200 g at RT for 15 minutes. The upper layeredplatelet-rich plasma (PRP) was collected, and centrifuged at 1000 g for10 minutes after mixing with anticoagulant (the final concentration: 0.5μM prostacyclin and 10 U/ml heparin). The supernatant was removed, andthe platelet pellets were resuspended in Tyrode's solution and furthercentrifuged at 1000 g for 10 minutes. Finally, the wash plateletswithout plasma proteins were resuspended in the Tyrode's solutioncontaining calcium and magnesium ions (this sample is wash platelets).The number of platelets were calculated using the coulter counter beforeuse, and the density of platelets was adjusted to 3×10⁸ cells/ml andstored at RT for use.

Experiment 2: Human Platelet Aggregation test

Experiment 2 was performed to determine the variations of lighttransmission upon the aggregation of platelets (the platelet-rich plasmasample and the wash platelet sample) by using platelet aggregometer(Model 570VS, Chrono-log Corp., U.S.). Firstly, the platelets (3×10⁸cells/ml) prepared in Experiment 1 was pre-heated with stir at 900 rpmat 37° C. for 1 minute, and the prepared 5-nitrobenzoate derivative(compound 4 or other control compounds 5, 6 and 7) was added to reactfor 3 minutes. The separate platelet activation stimulator (includes butnot limit to ADP, collagen, U46619, thrombin and A23187) was added toobserve the effect of 5-nitrobenzoate derivative on the plateletaggregation activation.

Please refer to FIGS. 1( a), 1(b), 1(c), 1(d) and 1(e), whichrespectively depict the effect of compound 4 or control compound 5 on(a) ADP-, (b) collagen-, (c) U46619-, (d) thrombin- and (e)A23187-induced wash platelet aggregation test. Compound 4 did notinhibit or interfere ADP-, collagen-, U46619-, thrombin- orA23187-induced platelet aggregation along with the increased dosage ofcompound 4.

Please refer to FIG. 2( a), which depicts the effect of compound 4 andcontrol compounds 5 to 7 on the ADP-induced platelet-rich plasmaaggregation test. The measured light transmission of plateletaggregation was enhanced depending on the increased reaction time (after300 seconds) of compound 4 (group 2), indicating that compound 4 did notinhibit or interfere ADP-induced platelet-rich plasma aggregation.Please refer to FIG. 2( b), similarly, the measure light transmission ofplatelet-rich plasma aggregation test was enhanced depending on theincreased reaction time (after 300 seconds) of compound 4 (group 2),indicating that compound 4 did not inhibit or interfere collagen-inducedplatelet aggregation.

Experiment 3: Tumor Cell-Induced Platelet Aggregation (TCIPA)

The purified platelets (1×10⁹ cells/ml) was preheated with stir at 900rpm at 37° C. for 1 minute, and 5-nitrobenzoate derivative of theinvention was added. After a 3-minute reaction, C6 tumor cells “C6-Lung”and “C6-LG” (1×10⁶ cells/ml, respectively) with different levels ofpodoplanin was added to react with platelets for 15 minutes, and thevariations of light transmission upon the platelet aggregation weremeasured by using platelet aggregometer, to analyze the TCIPA effect.

Please refer to the immunoblotting pattern in FIG. 3( a), which depictsthat C6-Lung tumor cells had the higher expression level of podoplaninrelative to C6-LG or C6-Blood cells. β-Actin is the control forimmunoblotting test.

Please refer to FIG. 3( b), which depicts that compound 4 (20 μM) caneffectively inhibit C6-Lung tumor cell (with high expression level ofpodoplanin)-induced platelet aggregation along with the increasedreaction time.

Experiment 4: Platelet Aggregation Induced by the RecombinantPodoplanin/Fc Fusion Protein

The purified wash platelets (1×10⁹ cells/ml) were preheated with stir at1000 rpm at 37° C. for 1 minute, and 5-nitrobenzoate derivative offormula I of the invention was added. After a 3-minute reaction, thegenetically engineering recombinant podoplanin/Fc fusion protein(abbreviated hereinafter “PDPN/Fc”, 2 μg, Sino Biological Inc., Beijing,People's Republic of China) was added to react with platelets for 15minutes, and the variations of light transmission upon the plateletaggregation were measured by using platelet aggregometer, to analyze theeffect of 5-nitrobenzoate derivative on the recombinant PDPN/Fc-inducedplatelet aggregation.

Please refer to FIG. 4, which depicts that compound 4 can effectivelyinhibit the PDPN/Fc-induced platelet aggregation along with theincreased reaction time. The recombinant Fc is the geneticallyengineering antibody Fc fragment and acts as the control.

In concluding the above experimental results, 5-nitrobenzoatederivatives or compounds of Formula I with mono-substituted benzoylchloride, di-substituted benzoyl chloride or tri-substituted benzoylchloride, of the invention do not influence platelet aggregation, canefficiently inhibit tumor cell-induced platelet aggregation (TCIPA) andthe TCIPA pathway, can specifically inhibit podoplanin-induced plateletaggregation and its pathway, in particular inhibit the recombinantpodoplanin/fc fusion protein-induced platelet aggregation

Since podoplanin of tumor cells would be combined with CLEC-2 ofplatelets and 5-nitrobenzoate derivatives of the invention would inhibitTCIPA induced by podoplanin-expressing tumor cells, 5-nitrobenzoatederivatives of the invention can be used to block the interactionbetween CLEC-2 and podoplanin and can be applied as the targeted therapymedicine for inhibiting metastasis of tumor cells.

While the invention has been described in terms of what is presentlyconsidered to be the most practical and preferred Embodiments, it is tobe understood that the invention needs not be limited to the disclosedEmbodiments. On the contrary, it is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the appended claims, which are to be accorded with the broadestinterpretation so as to encompass all such modifications and similarstructures.

What is claimed is:
 1. A method of inhibiting metastasis of a tumor, themethod comprising a step of administrating to a subject in need thereofan effective amount of a 5-nitrobenzoate derivative as represented byformula I,

wherein R one is selected from a group consisting of a hydrogen, anunsubstituted benzoyl moiety

a mono-substituted benzoyl moiety

a di-substituted benzoyl moiety

and a tri-substituted benzoyl moiety

and each of R₁, R₂, R₃, R₄, R₅ and R₆ is one selected from a groupconsisting of a fluoride, a chloride, a bromide, an iodide and a methylgroup.
 2. The method according to claim 1, wherein the subject is one ofa human and a mammal excluding the human.
 3. A method for blocking aninteraction between a C-type lectin-like receptor 2 and a podoplanin orblocking an effect caused by the interaction therebetween, by comprisingstep of using a 5-nitrobenzoate derivative as represented by formula I,

wherein R is one selected from a group consisting of a hydrogen, anunsubstituted benzoyl moiety

a mono-substituted benzoyl moiety (

a di-substituted benzoyl moiety

and a tri-substituted benzoyl moiety

and each of R₁, R₂, R₃, R₄, R₅ and R₆ is one selected from a groupconsisting of a fluoride, a chloride, a bromide, an iodide and a methylgroup.
 4. A method for blocking a pathway of a tumor cell-inducedplatelet aggregation, comprising a step of using a 5-nitrobenzoatederivative as represented by formula I,

wherein R is one selected from a group consisting of a hydrogen, anunsubstituted benzoyl moiety

a mono-substituted benzoyl moiety (

a di-substituted benzoyl moiety

and a tri-substituted benzoyl moiety

and each of R₁, R₂, R₃, R₄, R₅ and R₆ is one selected from a groupconsisting of a fluoride, a chloride, a bromide, an iodide and a methylgroup.
 5. A 5-nitrobenzoate derivative represented by formula I,

wherein R is one selected from a group consisting of a hydrogen, anunsubstituted benzoyl moiety

a mono-substituted benzoyl moiety (

a di-substituted benzoyl moiety

and a tri-substituted benzoyl moiety

each of R₁, R₂, R₃, R₄, R₅ and R₆ is one selected from a groupconsisting of a fluoride, a chloride, a bromide, an iodide and a methylgroup.
 6. The 5-nitrobenzoate derivative according to claim 5 beingprepared as a pharmaceutical composition.
 7. The 5-nitrobenzoatederivative according to claim 5 being prepared as a pharmacologicallyacceptable salt.
 8. A method for preparing a 5-nitrobenzoate derivativeas represented by formula I,

the method comprising a step of using anN-(2-aminoethyl)-2-hydroxy-5-nitrobenzamide as one of a startingmaterial and an intermediate, wherein R is one selected from a groupconsisting of a hydrogen, an unsubstituted benzoyl moiety (

a mono-substituted benzoyl moiety

a di-substituted benzoyl moiety

and a tri-substituted benzoyl moiety

and each of R₁, R₂, R₃, R₄, R₅ and R₆ is one selected from a groupconsisting of a fluoride, a chloride, a bromide, an iodide and a methylgroup.